Enzyme immobilization concerns immobilizing an enzyme product on a carrier on which the enzyme is fixed and yet functional and for which the enzyme is not liberated to the solvent to which it is applied. The most commonly immobilized enzymes are glucose isomerase used for isomerization reactions.
The industrial use of enzymes is often limited by their high cost and rapid inactivation. To improve their economic feasibility in industrial processes, enzymes are generally immobilized onto a matrix. Immobilization facilitates re-use of the enzymes, and may affect the selectivity and stability of the enzyme. Immobilization research has mainly focused upon means to enhance the transfer of enzymes onto the support, and upon means to ensure that the transferred enzymes remain active.
A number of different organic and inorganic support matrices and enzyme immobilization techniques have been tried with a view to achieve a high level of enzyme uptake with a minimum of enzyme degradation or inactivation.
A widely used approach to enzyme immobilization might be referred to as the covalent cross-linking process and is exemplified by U.S. Pat. No. 4,071,409 (Messing et al.). According to the teaching of this patent a support medium is modified or coated to present functionalities which can then be linked by way of a cross-linking agent to free functional groups of the enzyme.
In many industrial immobilization processes described in the prior art, the carrier or support material is placed in a column shaped adsorption vessel and an enzyme containing liquid is recirculated until sufficient adsorption of the enzyme on the carrier has been obtained. Following the adsorption step the column is emptied by manually shoveling the enzyme-carrier product into trays. The product is then dried by placing the trays under vacuum at room temperature for a period of 14-16 hours.
EP 0216272 describes a granular diatomaceous earth which is treated with a polyamine and reacted with e.g. glutaraldehyde, where after it is reacted with enzyme to form an immobilized enzyme. It is prepared in aqueous solution in a columnar reactor.
EP 0641859 describes a granular diatomaceous earth carrier which is treated with a polyamine. Further an amine reactive material is reacted with enzyme which is contacted with the carrier to form an immobilized enzyme. It is prepared in aqueous solution in a column.
U.S. Pat. No. 4,438,196 describes a carbon carrier which is reacted with a polyamine, the carrier is further reacted with a reactant and finally the enzyme to form an immobilized enzyme.
U.S. Pat. No. 4,141,857 describes the preparation of an enzyme support which is prepared by reacting a porous carrier with a polyamine and thereafter a reactant.
Other immobilization processes are described in WO 95/22606 (Pedersen et al.) and WO 99/33964 (Christensen et al.).
WO 95/22606 describes a process, wherein an enzyme containing liquid is brought in contact with porous silica carrier in an extruder or a granulation apparatus.
WO 99/33964 discloses an immobilization process wherein the immobilization is prepared in a fluid bed apparatus.
CA 2277371 describes a process for immobilization of an enzyme by incubating a siliceous support having surface hydroxyl groups with a first aqueous solution comprising a polyaldehyde and subsequently allowing a second aqueous solution comprising an enzyme to come into contact with the modified support and finally removing the support from the solution.
EP 133531 describes a process for immobilisation of an enzyme by (a) introducing into an aqueous medium containing an enzyme and a polyethyleneimine and (b) adding of glutaraldehyde and chitosan to the aqueous medium and subsequently removing the cross linked product from the liquid medium.
In U.S. Pat. No. 4,888,285 (Nishimura et al.) a silica gel is modified by reaction with an aminosilane derivative in an organic solvent. The resulting aminated support is then linked to the enzyme in the presence of glutaraldehyde, tannic acid and chitosan.
EP 0206687 discloses an immobilization process comprising mixing a dispersion of enzyme with polyazetidine prepolymer and glutaraldehyde followed by dewatering.
Immobilized enzymes are known to be used in continuous enzymatic reactions within a variety of industrial applications, including waste water treatment, production of pharmaceuticals and chemicals.